Using mating-type loci to improve taxonomy of the Tuber indicum complex, and discovery of a new species, T. longispinosum

Black truffles that morphologically resemble Tuber indicum have been known to occur in Japan since 1979. Our previous studies showed that there are two phylotypes of these truffles, both of which fell into the T. indicum complex (hereinafter "Tuber sp. 6" and "Tuber sp. 7"). However, their taxonomic treatment is still unclear. In this study, we conducted morphological and phylogenetic analyses for a total of 52 specimens from Japan (16 Tuber sp. 6 and 13 Tuber sp. 7), China (10 T. himalayense and 8 T. indicum), and Taiwan (5 T. formosanum). We compared ascospore ornamentation, size, distribution of asci with average number of spores per ascus, spine size and shape of the Japanese specimens with their allied taxa. For phylogenetic analysis, we sequenced two mating loci (MAT1-1-1 and MAT1-2-1) and three commonly used loci (ITS, β-tubulin, and TEF1-α). Three distinct lineages were recognized by phylogenetic analyses based on the sequences of the two mating-related loci and three independent loci. The Tuber sp. 6 sequences clustered with those of T. himalayense and T. formosanum, and there was no clear difference in morphology among them. Tuber sp. 7 formed a distinct lineage in each phylogram. The specimens tended to have five-spored asci more frequently than other allied species and could be characterized as having ascospore ornamentation with longer spines and narrower spine bases. We therefore described Tuber sp. 7 as a new species (T. longispinosum), and treat Tuber sp. 6 and T. formosanum as synonyms of T. himalayense.